Abstract
BackgroundRecent advances in molecular genetic analysis using next‐generation sequencing (NGS) have drastically accelerated the identification of disease‐causing gene mutations. Most next‐generation sequencing analyses of inherited diseases have mainly focused on single‐nucleotide variants and short indels, although, recently, structure variations including copy number variations have come to be considered an important cause of many different diseases. However, only a limited number of tools are available for multiplex PCR‐based target genome enrichment.MethodsIn this paper, we reported a simple and efficient copy number variation visualization method for Ion AmpliSeq™ target resequencing data. Unlike the hybridization capture‐based target genome enrichment system, Ion AmpliSeq™ reads are multiplex PCR products, and each read generated by the same amplicon is quite uniform in length and position. Based on this feature, the depth of coverage information for each amplicon included in the barcode/amplicon coverage matrix file was used for copy number detection analysis. We also performed copy number analysis to investigate the utility of this method through the use of positive controls and a large Japanese hearing loss cohort.ResultsUsing this method, we successfully confirmed previously reported copy number loss cases involving the STRC gene and copy number gain in trisomy 21 cases. We also performed copy number analysis of a large Japanese hearing loss cohort (2,475 patients) and identified many gene copy number variants. The most prevalent copy number variation was STRC gene copy number loss, with 129 patients carrying this copy number variation.ConclusionOur copy number visualization method for Ion AmpliSeq™ data can be utilized in efficient copy number analysis for the comparison of a large number of samples. This method is simple and requires only easy calculations using standard spread sheet software.
Highlights
Recent advances in molecular genetic analysis using nextgeneration sequencing (NGS) have drastically accelerated the identification of disease-causing gene mutations in a relatively short period
We describe an efficient copy number variations (CNVs) visualization method for Ion AmpliSeq data, which can be utilized in efficient CNV analysis for the comparison of a large number of samples
To assess the utility of this method, we analyzed three previously reported Japanese hearing loss patients with STRC gene copy loss identified from the results of next-generation sequencing analysis with hybridization capture-based target genome enrichment, and we confirmed this CNV by high-resolution array comparative genomic hybridization (Moteki et al, 2016)
Summary
Department of Otorhinolaryngology, Shinshu University School of Medicine, Matsumoto City, Japan Funding Information This study was aided by Health, Labour and Welfare of Japan Scientific Research Grants (H25-Kankaku-Ippan-002, H26Nanchitou(Nan)-Ippan-032), grants from the Japan Agency for Medical Research and Development (JP17ek0109114, JP17kk0205010), and a Grant-in-Aid for Scientific Research (A) (15H02565) from the Ministry of Education, Science and Culture of Japan. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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